Weaver Mouse Cerebellum Research Articles

Expression of GluR6 kainate receptor subunit in granular layer of weaver mouse cerebellum

The Girk2 ( wv ) (weaver) mutation impairs migration of cerebellar granule cells from external to internal granular layer and induces neuronal death during the first 2 weeks of postnatal life. Kainate receptors are heteromeric ionotropic receptors of glutamate consisting of five subunits termed GluR5, GluR6, GluR7, KA1 and KA2. In order to investigate whether the weaver gene affects the expression of kainate receptors in weaver cerebellum, we determined mRNA expression levels of GluR6 kainate receptor subunit and [(3)H]kainic acid specific binding in the developing cerebellum, using in situ hybridization and receptor film autoradiography, respectively. In the weaver postnatal day 10 (P10) cerebellum, our data indicated lower levels of GluR6 mRNA expression and lower [(3)H]kainic acid specific binding in external granular layer (EGL) compared to normal EGL. Our results are indicative of either down-regulation of kainate receptors or modulation of their functional characteristics in weaver granule cells.

Purkinje cell age-distribution in fissures and in foliar crowns: a comparative study in the weaver cerebellum

Generation and settling of Purkinje cells (PCs) are investigated in the weaver mouse cerebellum in order to determine possible relationships with the fissuration pattern. Tritiated thymidine was supplied to pregnant females at the time that these neurons were being produced. Autoradiography was then applied on brain sections obtained from control and weaver offspring at postnatal (P) day 90. This makes it possible to assess the differential survival of neurons born at distinct embryonic times on the basis of the proportion of labeled cells located at the two foliar compartments: fissures and foliar crowns. Our data show that throughout the surface contour of the vermal lobes, generative programs of PCs were close between wild type and homozygous weaver. Similar data were found in the lobules of the lateral hemisphere. On the other hand, the loss of PCs in weaver cerebella can be related to foliar concavities or convexities depending on the vermal lobe or the hemispheric lobule studied. Lastly, we have obtained evidence that late-generated PCs of both normal and mutant mice were preferentially located in fissures. These quantitative relationships lead us to propose a model in which the final distribution of PCs through the vermal contour would be coupled to two factors: the cortical fissuration patterning and a "time-sequential effect" of weaver mutation.

Trace elements monitored with neutron activation analysis during neurodegeneration in brains of mutant mice

Our objective was to test the efficacy of neutron activation analysis (NAA) for monitoring trace elements in discrete brain regions of wild-type and mutant mice (weaver)with well-documented spatiotemporal patterns of neuronal loss in the cerebellum. As neuronal degradation is accompanied by upregulation of metalloenzymatic activity, we used NAA to detect any transient elevation in elemental composition of brain tissue. Zinc and copper concentrations in the cerebellum of weaver mice aged 5 days greatly exceed levels observed in normal mice or in the weaver's forebrain, which is unaffected by neurodegeneration. Furthermore, concentrations of copper and zinc follow the spatiotemporal pattern of neurodegeneration observed in the cerebellum of weaver mutant mice.

G protein-activated inwardly rectifying K + channel inhibition and rescue of weaver mouse motor functions by antidepressants

Antidepressants, including tricyclic antidepressants (TCAs) and selective serotonin reuptake inhibitors (SSRIs), have been widely used for the treatment of not only depression but also other psychiatric disorders, although the molecular mechanisms of the drug effects have not yet been sufficiently revealed. Here, we investigated the in vivo effects of these antidepressants on G protein-activated inwardly rectifying K + (GIRK) channels, which are important for regulating the excitability of various cells, by using weaver ( w v ) mice, which have mutant GIRK channels and show abnormal neuronal cell death and motor disturbances. First, we found that a widely used SSRI fluoxetine (also known as Prozac) effectively inhibited w v GIRK2 channels like wild-type GIRK channels, expressed in Xenopus oocytes. Next, we found that weaver motor disturbances were remarkably alleviated by chronic treatment with fluoxetine or desipramine. Furthermore, the chronic fluoxetine treatment substantially suppressed the abnormal neuronal cell death in the weaver mouse cerebellum and pontine nuclei. These results suggest that continuous inhibition of w v GIRK2 channels by a group of antidepressants caused substantial suppression of the neuronal cell death and resulted in improvement of motor abilities in weaver mice. These results provide evidence for in vivo GIRK channel inhibition by a group of antidepressants.

Cell death in weaver mouse cerebellum.

Mice with the weaver mutation exhibit an uneven weave to their gait, ataxia, mild locomotor hyperactivity and, occasionally, tonic-clonic seizures. A single amino acid mutation in a G-protein coupled, inwardly rectifying K+ channel, GIRK2, gives rise to the symptoms seen in the weaver mice. Two areas of the brain are primarily affected. Cerebellar granule cell neurons die soon after birth and dopaminergic neurons are severely depleted in the substantia nigra. In this article we review recent studies of wild-type and mutant GIRK channels found in native cells or introduced into expression systems. We also review two models that explain some of the details leading to the neuronal cell death observed in weaver mice.

A Cell Cycle Alteration Precedes Apoptosis of Granule Cell Precursors in the weaver Mouse Cerebellum

A missense mutation in the gene coding for the G-protein-activated inwardly rectifying potassium (GIRK) channel, GIRK2, is responsible for apoptosis in the external germinal layer (EGL) of the cerebellum and a nonapoptotic death of midbrain dopaminergic neurons in the weaver (wv) mouse. Failure of axonogenesis and migration are considered to be the primary consequences of GIRK2 channel malfunction in the cerebellum. We investigated whether a disruption of the cell cycle precedes the failure of migration and axonogenesis and leads to massive apoptosis. To this end, immunohistochemistry and immunoblotting for PCNA, Cdk4, cyclin D, cyclin A, and the Cdk inhibitor p27/kip1, as well as in situ end-labeling for apoptotic DNA fragmentation, were applied to cerebella of P7-P21+/+, wv/+, and wv/wv mice. In +/+ and wv/+ mice, the expression of cell cycle proteins was limited to the outer, premigratory zone of the EGL. Antibodies to p27, a marker of cell differentiation, gave a reverse staining pattern. Due to migration delay, patches of p27-positive cells persisted in the outer EGL in P21 wv/+ mice. On the contrary, marked cell cycle up-regulation and absence of p27 occurred throughout the EGL at all ages in wv/wv mice, indicating an inability to switch off the cell cycle. Mitotic index evaluation showed that cell cycle activation was unrelated to proliferative events. Cell cycle proteins were not expressed in the substantia nigra, suggesting that nonapoptotic death of mature dopaminergic neurons is not preceded by abortive cell cycle re-entry. Our data show that abnormalities of the cell cycle in wv/wv cerebellum represent a major and early consequence of GIRK2 channel malfunction and may strongly influence the susceptibility of EGL cells to apoptosis. These observations may help in understanding the pathogenesis of human neurological channelopathies.

Weaver cerebellar granule neurons show altered expression of NMDA receptor subunits bothin vivo andin vitro

Biochemical, immunocytochemical, and molecular biological techniques were used to investigate the expression of N-methyl-D-aspartate (NMDA) receptor subunits in migration-deficient weaver mouse cerebellum in vivo and in primary cultures of the vermal weaver granule neurons with or without a rescue by verapamil. We found that both NMDAR1(zeta1) message and protein were expressed by the weaver granule neurons in situ. Immunocytochemical and biochemical analyses indicated that granule neurons of the weaver cerebellum expressed R1(zeta1) and R2A(epsilon1) subunits but showed little expression of the R2B(epsilon2) subunit. In weaver cerebellum, the R2B(epsilon2) subunit was primarily expressed in nerve fibers of the internal granule cell layer and white matter. Reverse-transcriptase-polymerase chain reaction followed by sequence analysis of the R1(zeta1) subunit indicated that the zeta1 subunit amplicons of both normal and weaver cerebella were identical, and that splice variants with exon 22 (1-2) and with or without exon 5 (a/b) or exon 21 (1-4) were detectable. The R2A(epsilon1), and R2B(epsilon2) subunits of the normal and weaver mouse cerebellum revealed no primary structural differences between the normal and weaver NMDA receptor subunits or the cloned mouse NMDA receptor subunits. In vermal cultures, normal granule neurons expressed all three NMDA receptor subunits (zeta1, epsilon1, and epsilon2), whereas the weaver neurons failed to express the epsilon2 subunit. Rescue of the weaver neurons by verapamil induced expression of the epsilon2 protein along the granule neuronal surfaces. The present results suggest that lack of the epsilon2 subunit in the weaver cerebellum may relate to the lack of functional NMDA receptors and/or to the migratory failure of the weaver granule neurons. Our data further suggest that NMDA receptor-mediated neurotoxicity is an unlikely mediator of neuronal death of the weaver granule neurons. In fact, down-regulation of the NMDA receptor expression and function may be a protective measure of the weaver granule neurons to reduce calcium entry via these receptors.

Weaver cerebellar granule neurons show altered expression of NMDA receptor subunits both in vivo and in vitro

Biochemical, immunocytochemical, and molecular biological techniques were used to investigate the expression of N-methyl-D-aspartate (NMDA) receptor subunits in migration-deficient weaver mouse cerebellum in vivo and in primary cultures of the vermal weaver granule neurons with or without a rescue by verapamil. We found that both NMDAR1(ζ1) message and protein were expressed by the weaver granule neurons in situ. Immunocytochemical and biochemical analyses indicated that granule neurons of the weaver cerebellum expressed R1(ζ1) and R2A(ϵ1) subunits but showed little expression of the R2B(ϵ2) subunit. In weaver cerebellum, the R2B(ϵ2) subunit was primarily expressed in nerve fibers of the internal granule cell layer and white matter. Reverse-transcriptase–polymerase chain reaction followed by sequence analysis of the R1(ζ1) subunit indicated that the ζ1 subunit amplicons of both normal and weaver cerebella were identical, and that splice variants with exon 22 (1-2) and with or without exon 5 (a/b) or exon 21 (1-4) were detectable. The R2A(ϵ1), and R2B(ϵ2) subunits of the normal and weaver mouse cerebellum revealed no primary structural differences between the normal and weaver NMDA receptor subunits or the cloned mouse NMDA receptor subunits. In vermal cultures, normal granule neurons expressed all three NMDA receptor subunits (ζ1, ϵ1, and ϵ2), whereas the weaver neurons failed to express the ϵ2 subunit. Rescue of the weaver neurons by verapamil induced expression of the ϵ2 protein along the granule neuronal surfaces. The present results suggest that lack of the ϵ2 subunit in the weaver cerebellum may relate to the lack of functional NMDA receptors and/or to the migratory failure of the weaver granule neurons. Our data further suggest that NMDA receptor–mediated neurotoxicity is an unlikely mediator of neuronal death of the weaver granule neurons. In fact, down-regulation of the NMDA receptor expression and function may be a protective measure of the weaver granule neurons to reduce calcium entry via these receptors. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 441–454, 1999

Bcl-2, Bax and Bcl-x expression in neuronal apoptosis: a study of mutant weaver and lurcher mice.

We investigated the expression of the apoptosis modulating proteins Bcl-2, Bax and Bcl-x in the cerebellum of mutant lurcher and weaver mice. Lurcher Purkinje cells and weaver germinal (granule neuron progenitor) cells both die via apoptosis during the postnatal cerebellar development. No significant changes in the expression patterns were detected prior to the actual cell death process. Instead apoptotic lurcher Purkinje cells exhibited increased Bax and Bcl-x expression, while surviving cells had an expression pattern similar to that of healthy littermates. Increased Bax expression was also found in apoptotic weaver germinal cells, while no change of Bcl-x expression was detected. Bcl-2 was expressed at low levels in cerebellar neurons and no loss of Bcl-2 was evident. The observed expression patterns of Bcl-2, Bax and Bcl-x protein in apoptotic lurcher and weaver neurons support the hypothesis that the execution of neuronal apoptosis involves increased expression of Bax, which could represent a general mechanism in diverse neurodegenerative processes.

Expression of neurotrophins and neurotrophin receptors in the cerebellum of mutant weaver and lurcher mice

To test the hypothesis whether a failure to express neurotrophins or a neurotrophin receptor might underlie the pathology observed in mutant mice with degeneration of regionally distinct subpopulations of neurons, the expression of BDNF, NT-3, TrkB, TrkC and synaptophysin mRNA was examined in the cerebellum of mutant lurcher ( lc/+) and weaver ( wv/+)/( wv/ wv) mice. To identify the expression patterns of individual neurons, we used in situ hybridization with digoxigenin labeled ribonucleotide probes. RT-PCR of cerebellar mRNA for BDNF, NT-3, TrkB and TrkC (GAPDH as internal standard) was performed in parallel. Although especially in homozygous ( wv/ wv) weaver mice the normal anatomical order and number of the cerebellar neurons is grossly disturbed, residual Purkinje and granule neurons of both mutants displayed a normal expression pattern of the neurotrophins examined. Thus, the affected animals showed no significant signal decrease compared to healthy littermates or C3H mice. Our results suggest that the loss of specific neuron populations in the cerebellum of either mutant occurs via mechanisms either independent or downstream of the neurotrophins examined in this study.

Regionalization defects in the weaver mouse cerebellum

Regionalization defects in the weaver mouse cerebellum

Regionalization defects in theweaver mouse cerebellum

The mammalian cerebellum consists of parasagittal bands and transverse zones that are laid down early in development. When the adult cerebellum is immunostained for the Purkinje cell-specific antigen zebrin II (i.e., aldolase C), compartmentation is reflected in alternating zebrin II+ (P+) and zebrin II- bands (P ). The zebrin II phenotype is Purkinje cell autonomous; thus, disruptions in the zebrin pattern may reflect early problems in pattern formation. Zebrin II expression has been examined in the weaver (wv) mouse cerebellum. Both zebrin II- and zebrin II Purkinje cells are present in the homozygous weaver (wv/wv) mouse, but they are not distributed normally. In the posterior vermis, although the zebrin II+ bands are wider and multilaminate, the standard compartmentation is present. However, a large zebrin II+ cell mass is absent from the central vermis, and analysis of the anterior lobe reveals several missing zebrin II- bands. The cytoarchitectonic defects in wv mice are not simply related to the Purkinje cell abnormalities. Instead, serial reconstruction reveals two transverse boundaries-one rostrally in lobule VI and the other caudally in lobule IX-that delineate cytoarchitectonic transverse zones important in cerebellar development. The abnormal zebrin expression pattern in wv/wv mice may be secondary to the deletion of a transverse zone. This is the first demonstration that Purkinje cell compartmentation can be altered by mutation; therefore, the wv mutation should prove valuable in understanding cerebellar regionalization.

MAP2 and GAP-43 expression in normal and weaver mouse cerebellum: correlative immunohistochemical and in situ hybridization studies.

MAP2 is a major microtubule-associated brain protein, selectively localized in dendrites; growth-associated phosphoprotein GAP-43 is a neuron-specific protein associated with axonal outgrowth. In adult cerebellum, both of these proteins and their corresponding RNA transcripts are most strongly expressed by granule cells. Using immunocytochemistry with antibodies and in situ hybridization histochemistry with [32P] labeled oligonucleotide probes, we examined the cellular localization of MAP2, GAP-43 and their mRNAs in the cerebellum of control and weaver (wv/wv) mutant mice, which exhibit massive granule cell death. In wild-type (+/+) mice, MAP2 immunoreactivity was seen in neuronal somata and dendrites of the granule cell layer; GAP-43 immunoreactivity was present in molecular layer, corresponding to the distribution of parallel fibres. Transcripts encoding MAP2 and GAP-43 were localized in the layer of the granule cell somata. In heterozygous weaver mice (wv/+), which feature an intermediate degree of granule cell loss, MAP2 immunoreactivity was localized in the granular layer, and the pattern of GAP-43 immunostaining was also similar to +/+, the only difference being a thinner molecular layer. Heterozygotes had an anatomical pattern of MAP2 and GAP-43 mRNA hybridization qualitatively similar to that of the wild-type with some deviations in signal intensity. In homozygous weaver mutants (wv/wv), MAP2 immunoreactivity was extremely weak in the area beneath Purkinje cells and a certain GAP-43 immunoreactivity was seen in the upper part of cerebellar cortex. Hybridization signals for MAP2 and GAP-43 mRNAs were minimal. The reported alterations in regional pattern of MAP2 and GAP-43 expression in mutant mice offer a molecular correlate of dendritic and axonal protein gene transcription pertinent to the neuropathological manifestations of certain forms of heredodegenerative ataxia.

BAPTA-AM and ethanol protect cerebellar granule neurons from the destructive effect of the weaver gene.

The mechanisms by which the weaver gene (Reeves et al., 1989; Patil et al., 1995) inhibits neurite extension and/or induces death of the granule neurons in homozygous weaver mouse cerebellum are not presently understood. Here we show that BAPTA-AM and ethanol, which either reduce cytosolic levels of free calcium or prevent calcium entry, promote neurite outgrowth of the weaver neurons similar to the L-type calcium channel blocker verapamil (Liesi and Wright, 1996). Importantly, BAPTA-AM, ethanol, and verapamil not only restore neurite outgrowth of the weaver neurons but adjust their depolarized resting membrane potentials to the levels of normal neurons. These results indicate that calcium-dependent mechanisms mediate the action of the weaver gene and that the weaver neurons can be normalized by blocking this calcium effect. We further report that BAPTA-AM and verapamil also have a neuroprotective effect on normal neurons exposed to high concentrations of ethanol. We suggest that verapamil should be evaluated as a drug for treatment of alcohol-induced brain damage and neurodegenerative disorders.

Ethanol-exposed central neurons fail to migrate and undergo apoptosis.

Prenatal exposure of human brain to ethanol impairs neuronal migration and differentiation and causes mental retardation. The present results indicate that the adverse effects of ethanol on brain development may be partly due to the ethanol-induced disturbance of neuronal interaction with laminin, a protein involved in neuronal migration and axon guidance. This report shows that physiological concentrations (IC50 = 28 mM) of ethanol inhibit neurite outgrowth and neuronal migration of the rat cerebellar granule neurons on a laminin substratum. The ethanol-treated granule neurons undergo apoptosis, degrade their laminin substratum, and appear to release and bind increased amounts of the B2-chain-derived peptides along their surfaces. A protease inhibitor aprotinin, and the NMDA receptor channel, and voltage-gated calcium channel antagonist MK801 partially protect cerebellar granule neurons from ethanol-induced neurotoxicity. These results imply that ethanol-treated granule neurons resemble the granule neurons of the homozygous weaver mouse cerebellum with respect to their apoptosis, laminin expression, and partial rescue by approtinin and MK-801. Thus, ethanol may influence neuronal survival and neurite outgrowth via molecular pathways similar to those involved in neuronal death in other neurodegenerative processes of the central nervous system.

Apoptotic cell death in the cerebellum of mutant weaver and lurcher mice

Apoptosis in the central nervous system of mutant weaver (wv) and lurcher (Ic) mice was studied using in situ DNA end-labeling. The number of apoptotic cells in the external granule cell layer of weaver (wv/+) mice at postnatal day 9 was increased six- to eight-fold compared to (+/+) littermates. Purkinje cells and Bergmann glia cells were not affected. No labeled cells were found in the substantia nigra. In lurcher (lc/+) mice, labeled nuclei of large cells were found in the Purkinje cell layer, suggesting that Purkinje cells of mutant lurcher mice undergo apoptosis. Apoptotic granule cell nuclei were found in the internal granule cell layer. In contrast to weaver (wv/+) mice, the number of apoptotic cells in the external granule cell layer was not increased. Apoptosis appears to be the common pathway of cell death in the degenerative processes induced by the weaver and the lurcher gene, respectively. These mutant mice offer the opportunity to study the mechanisms that underlie inappropriate apoptotic cell death in vivo.

Expression of the death-associated gene RP-8 in granule cell neurons undergoing postnatal cell death in the cerebellum of weaver mice

RP-8 is one of several mRNAs elevated during apoptosis in immature thymocytes. We used in situ hybridization to look for RP-8 mRNA in the cerebella of weaver mice. In the weaver mouse cerebellar granule cells fail to differentiate and instead die during the first two weeks of postnatal development [30]. We demonstrate that the dying cells exhibit DNA degradation, a characteristic feature of apoptosis. RP-8 is expressed in the weaver cerebellum during the period of granule cell death, and is limited to cells in the same region where apoptotic granule cells are located. Thus, RP-8 expression, first associated with PCD in thymocytes, correlates with granule cell death in the weaver cerebellum.

Increased Proteolytic Activity of the Granule Neurons May Contribute to Neuronal Death in the Weaver Mouse Cerebellum

The weaver mouse mutation is a genetic defect of unknown origin that leads to impairment of cerebellar granule neuronal migration and to neuronal cell death. We investigated laminin expression and proteolytic enzyme activity in this migration-deficient mouse mutant in vivo and in vitro to search for a molecular basis for the weaver defect. The weaver cerebellum showed a general increase in immunoreactivity for laminin, for a neurite outgrowth domain of the B2 chain of laminin, and for tissue plasminogen activator compared to the normal animals. Zymographic assays and immunocytochemistry confirmed that tissue plasminogen activator was the proteolytic enzyme synthesized in excess in the weaver mouse cerebellum in vivo. When placed in culture, the weaver granule neurons survived poorly on a laminin substratum, and failed to extend long neurites, unlike the normal cerebellar granule neurons. The cultured weaver granule neurons were proteolytically overactive and secreted excessive amounts of tissue plasminogen activator, which was likely to interfere with their neurite outgrowth potential on a laminin substratum. Indeed, the weaver granule neurons but not the normal neurons degraded laminin from their culture substratum and deposited a neurite outgrowth domain of the B2 chain of laminin onto their surfaces. Electrophysiology showed that the weaver granule neurons had poor resting membrane potentials (-38 V), whereas the normal neurons had normal resting membrane potentials of (-61 V). The resting membrane potentials of the weaver granule neurons were restored to near normal (-59 V) by a protease inhibitor, aprotinin. Aprotinin also rescued the weaver granule neurons from death on a laminin substratum and promoted their neurite outgrowth to the level of the normal animals. These results indicate that increased proteolytic activity accompanied with increased synthesis of laminin, and its B2 chain, distinguish the weaver mutation from the normal animals. These molecular changes may contribute to the impairment of granule neuronal migration and to the neuronal death, characteristic of the weaver mutation.

Development and death of external granular layer cells in the weaver mouse cerebellum: a quantitative study

Previous studies have identified the cerebellar granule cell as a primary site of gene action in the weaver mutant mouse. The temporal expression of the weaver mutant granule cell phenotype has not been fully investigated. To identify early postnatal expression of the weaver mutant phenotype, we quantitated 4 parameters of cerebellar development in postnatal day 0, 2, 4, 6, and 8 +/+, wv/+, and wv/wv mice: (1) cerebellar area, (2) cells in the external granule layer (EGL), (3) number of mitotic figures in the EGL, and (4) number of pyknotic figures in the EGL. Qualitative observations suggest a generalized retardation in the development of wv/wv and wv/+ EGL cells compared with the +/+ cells. Quantitatively, the earliest detectable mutant phenotype in wv/wv and wv/+ cerebella is the increased presence of cell death in the EGL, apparent at the day of birth. Degenerating cells in the EGL, the majority of which are found in the postmitotic zone of this layer, contain abnormally clumped heterochromatin, suggestive of "nuclear" cell death. Previous hypotheses concerning the mechanism of weaver gene action have suggested that granule cells die due to their inability to migrate from the EGL. The time course, mode, and spatial organization of cell death found in the present studies lead us to suggest that an earlier event in granule cell development, such as the exit of neuroblasts from the cell cycle or axonogenesis, is affected by the weaver gene.

Reorganization of cerebellar cell suspension transplanted into the weaver mutant cerebellum and immunohistochemical detection of synaptic formation

Dissociated cells prepared from the cerebellar primordia of normal 15-day mouse embryos were grafted into the cerebellum of 1-month-old weaver mutant mice which are characterized by degeneration of cerebellar granule cells during the early postnatal period. The growth of the grafted cells was investigated at 1 month after the operation. Implanted cells were highly developed to form a large mass of tissue in the host cerebellar folia. Histological examination revealed that a trilaminar cortical structure was partially developed in certain areas of the grafted tissue. The implanted granule-like cells were labeled with [ 3H]thymidine which was injected into the host, suggesting that the granule-like cells actively proliferate in the host cerebellum after the transplantation. In this area, strong immunoreactivity with synapsin I was detected indicating that the dissociated granule cells of the cerebellar primordia are able to develop a synaptic organization in the weaver mouse cerebellum.